Question

A cell-free method of amplifying DNA first developed in the mid 1980's revolutionised the field of biotechnology, Name the method and explain the basic steps of the technique involved.

Answer 1

In molecular biology, the Polymerase Chain Reaction (PCR) method is used to multiply a gene or a portion of DNA. It is heavily utilised during the gene-editing process. A primer, a template strand, and a thermostable DNA polymerase enzyme from the bacterium Thermus aquaticus are used in the in-vitro synthesis of sequences. The DNA segment is multiplied by a constant rate of DNA replication to a maximum of 1 billion copies.

1. Denaturation: The two strands of the double-stranded DNA molecules are split into a single strand by heating them to a high temperature (94° C). Denaturation is the term for this action. The DNA synthesis process uses each strand as a template.
2. Annealing: Since the sequences of the two oligonucleotide primers are complementary to the 3' ends of the template DNA, they anneal (hybridise) to each of the single-stranded DNA templates in this stage. Depending on the length and order of the primers, this step is performed at a lower temperature. The original DNA molecule is duplicated as a result.
3. Extension of primers: The primers are extended by DNA polymerase (Taq polymerase) using the nucleotides present in the process. 72°C is the ideal temperature for this polymerization phase. To acquire many copies of the rDNA segment, this procedure is performed several times. The amplified region can be ligated to a vector and used for further cloning. As a result, recombinant DNA is produced (rDNA).